Cryptosporidiosis is a gastrointestinal illness caused by the protozoan parasite Cryptosporidium species, which is a leading cause of diarrhea in a variety of vertebrate hosts. The primary mode of transmission is through oral routes; infections spread with the ingestion of oocysts by susceptible animals or humans. In humans, Cryptosporidium infections are commonly found in children and immunocompromised individuals. The small intestine is the most common primary site of infection in humans while extraintestinal cryptosporidiosis occurs in immunocompromised individuals affecting the biliary tract, lungs, or pancreas. Both innate and adaptive immune responses play a critical role in parasite clearance as evident from studies with experimental infection in mice. However, the cellular immune responses induced during human infections are poorly understood. In this article, we review the currently available information with regard to epidemiology, diagnosis, therapeutic interventions, and strategies being used to control cryptosporidiosis infection. Since cryptosporidiosis may spread through zoonotic mode, we emphasis on more epidemiological surveillance-based studies in developing countries with poor sanitation and hygiene. These epidemiological surveys must incorporate fecal source tracking measures to identify animal and human populations contributing significantly to the fecal burden in the community, as mitigation measures differ by host type.

Cryptosporidiosis is a gastrointestinal illness caused by Cryptosporidium species, which is transmitted most often by direct fecal contamination or through waterborne routes and cause diarrhea in a variety of vertebrate hosts. In humans, Cryptosporidium infection is commonly found in children and immunocompromised individuals. In children, cryptosporidiosis has been associated with impairment in growth, physical fitness, and cognitive function and has been identified as the leading global cause of diarrheal mortality among infants aged between 12 and 23 months.[1],[2] Cryptosporidiosis has emerged as the second major cause of diarrheal disease and death in infants as reported in the last few decades.[3] The first waterborne outbreak of Cryptosporidium was reported in Texas, USA in 1984.[4] A decade later, a major outbreak was documented in Milwaukee, Wisconsin, USA, affecting nearly 400,000 persons.[5] Recently, a major Cryptosporidium outbreak was reported in Sweden affecting 27,000 persons [6] and a foodborne outbreak of cryptosporidiosis was reported in the UK.[7] Since then, other outbreaks have been reported worldwide, highlighting the global significance of this parasite in [Figure 1].[8]

Figure 1: Major worldwide occurrence of human cryptosporidiosis outbreaks and sporadic cases: A color coded. distribution of major cases of cryptosporidiosis reported in different countries of the world between 1984-2013. Water-borne outbreaks represented with star symbol, round dot represents Food-borne outbreaks in the map, yellow color in the map represents the presence of cryptosporidiosis and white color represents no such reports are present

Cryptosporidiosis has great public health importance as it also poses occupational risks to the human population. For example, asymptomatic food workers infected with Cryptosporidium species can be the source of transmissions in the outbreaks.[9],[10],[11] Due to the public health concerns, the National Institutes of Health (NIH, USA) currently spends roughly US $4.3 million each year on carrying out basic research on Cryptosporidium, and apart from NIH, philanthropic organizations such as the Bill and Melinda Gates Foundation, are also focusing on interventional aspects for controlling Cryptosporidium infections.[3]

Cryptosporidiosis is usually a self-limiting illness in healthy individuals and lasts on average up to 9–15 days, while in immunocompromised individuals cryptosporidiosis can be life-threatening as there is no fully effective drug treatment. The small intestine is the primary site of Cryptosporidium infection in humans, and in immunocompromised individuals, the extraintestinal infectious may affect other organs such as the biliary tract, lungs, or pancreas.[12] Although cryptosporidiosis can be asymptomatic in some individuals, the most common clinical symptoms include profuse watery diarrhea, nausea, vomiting cramps such as abdominal pain and mild fever.[13],[14] A recent study also suggested that infection with Cryptosporidium parvum could lead to digestive carcinogenesis in humans.[15] In recent years, the molecular biology of Cryptosporidium has advanced significantly, leading to the development of novel approaches for detecting Cryptosporidium. It is now important to revisit the current problem of cryptosporidiosis and address updates in the biology, epidemiology, transmission, detection, and treatment, where techniques and approaches emerging in developed countries can be applied to improve health and livelihoods in threshold and developing country settings as well.

Epidemiology

Cryptosporidium spp. has a wide host range and can infect all four classes of vertebrates.[16],[17] To date, 27 species and more than 60 genotypes of Cryptosporidium have been identified worldwide.[18],[19] In the genus, Cryptosporidium, Cryptosporidium hominis, and C. parvum are the major etiological agents of human clinical cryptosporidiosis. In addition, a novel spp. Cryptosporidium viatorum was recently identified among travelers returning to Great Britain from the Indian subcontinent.[20] Ng-Hublin et al. reported the occurrence of a mixed infection of Cryptosporidium species – Cryptosporidium meleagridis, the Cryptosporidium mink genotype, and an unknown Cryptosporidium species in immunocompetent individuals.[21]

Cryptosporidium accounts for almost 20% of diarrheal episodes in children in developing countries, and up to 9% of diarrheal episodes in developed nations.[22] A high prevalence of Cryptosporidium infections in children has been reported from various countries.[23],[24],[25]Cryptosporidium infection has also been observed in immunocompromised individuals such as HIV-infected persons.[16],[17],[18],[19],[20],[21],[22],[23],[24],[25],[26] Several Cryptosporidium seroprevalence studies in humans are available that showed immunoglobulin G (IgG) response specifically to 15/17-kDa Cryptosporidium sporozoite antigen complex and the 27-kDa antigen.[27] One study from Europe provides serological evidence of Cryptosporidium infection in blood donors (83% positive), perhaps explaining the infrequent occurrence of clinically detectable cryptosporidiosis in the study region.[28]

Epidemiological studies have shown that the geographical distributions of Cryptosporidium spp. vary around the world. C. hominis is more prevalent in North and South America, Australia, and Africa, while C. parvum is localized more in Europe, especially in the UK.[29] One study from the UK suggests C. parvum is more common in rural populations mainly in the spring season while C. hominis is more in urban residents and peaks in late summer and autumn.[27] Waterborne and foodborne transmission of Cryptosporidium are the major routes of infection, although person to person contact, especially in day care settings and between men who have sex with men have also been reported.[30]Cryptosporidium organisms have been reported in sputum and respiratory tract of humans,[31],[32] thus suggesting a novel mode of respiratory transmission of cryptosporidiosis.

Various reports have also highlighted the presence of Cryptosporidium infection in large categories of vertebrate animals.[33],[34],[35] Serological responses against Cryptosporidium in animals also indicate the continuous exposure of this parasite in animals.[36],[37] One study from Uganda showed that Cryptosporidium has a prevalence rate of 11% in nonhuman primates and 2.2% in livestock,[38] while another study showed a prevalence of 6% in domestic and wild animals.[17] Few studies have also reported the existence of novel zoonotic Cryptosporidium species in fishes and giant panda.[39] Recently, Cryptosporidium ubiquitum has emerged as a zoonotic pathogen capable of causing cryptosporidiosis in humans.[40]

Cryptosporidium infection spreads with the ingestion of oocysts by susceptible animals or humans. [Figure 2] shows detailed description of transmission route in humans and animals. The incubation period of this protozoan parasite ranges from 1 to 2 weeks. Most patients with symptomatic infection present with acute watery diarrhea that lasts for a few days to 2 weeks, but sometimes, it can be persistent and last for up to 5 weeks. Cryptosporidiosis can become endemic in developing countries, where the hygiene and sanitation conditions are not adequate. As Cryptosporidium spreads through contaminated food under un-hygienic conditions, it is expected that all individuals in developing countries may be exposed at an early age. In a birth cohort study from India, the serum IgG response and seroconversion pattern to Cryptosporidium gp-15 among children suggested a high rate of asymptomatic transmission of Cryptosporidium in the study region.[41]

Cryptosporidium primarily infects the epithelium of small intestines but occasionally can infect lungs or bile ducts.[42],[43],[44] Complete genome sequences for C. parvum and C. hominis are now available.[45],[46] The Cryptosporidium genome consists of eight chromosomes containing nearly 9.2 million base pairs. Genome alignment studies showed that both of these genomes consist of 3%–5% of sequence variations with no significant insertion deletion or rearrangement.[47] Several metabolic pathways, multiple organelles, and genes, which are common to eukaryotes or restricted to Apicomplexa, are either reduced or missing in Cryptosporidium. Cryptosporidium also lacks the enzymes for the synthesis of key biochemical building block such as sugars, amino acids, and nucleotides. Genome analysis of Cryptosporidium shows that it encodes more than 80 genes with strong similarities to known transporters and several hundred genes with transporter-like properties. Therefore, Cryptosporidium spp. relies heavily on scavenging nutrients from the host, salvage rather than de novo biosynthesis, and glycolysis or substrate-level phosphorylation for energy production.

Cryptosporidium has a complex life cycle, consisting of both sexual and asexual phases. It completes its life cycle within a single host, i.e., humans. The infective stage of Cryptosporidium is the oocyst, each containing four sporozoites.[48] Oocysts are released into the environment after the defecation of an infected host and are later ingested by a susceptible host. These oocysts can remain viable in the environment and can resist standard disinfectants such as chlorination of drinking water.[49]Cryptosporidium is highly infectious, with ingestion of as few as ten oocysts by a healthy human resulting in clinical infection.[50],[51] After the ingestion of oocysts, spindle-shaped sporozoites are released into the gastrointestinal tract, which adhere to host intestinal epithelial cells (IECs). Encapsulation of sporozoites by parasitophorus vacuoles lead to the development of trophozoites. All stages of parasitic differentiation occur within the host cell plasma membrane. These trophozoites undergo merogony to produce meronts. Two consecutive generations of merogony occur, producing Type I and Type II meronts respectively. Type I merozoites, released from Type I meront, re-infect the nearby cells and thereby complete the asexual phase of reproduction.

The sexual phase of reproduction involves the Type II meronts, which differentiate into microgamonts (male gametes) and macrogamonts (female gametes). Macrogamonts, fertilized by the microgamonts, undergo successive division to form mature oocysts. As the result of sporogony two types of oocysts are formed, thick walled which are generally shed in the feces and thin walled which remain in the host intestine, and are responsible for autoinfection.

Several studies have elucidated various virulence mechanisms involved during sporozoite excystation, host cell adherence, invasion, intracellular maintenance, and host cell destruction occurring during Cryptosporidium infection.[52] A recent study suggests that C. parvum elongation factor 1α and novel Ca-activated apyrase plays an important role in host cell invasion.[53] Sporozoite apical protein also plays an important role in target cell attachment and parasitophorous vacuole formation.[54] Various host factors have also been implicated as critical determinants of the outcomes of host-pathogen interactions during Cryptosporidium infection.[55] The host immune response plays an important role in host-pathogen interactions by affecting both the probability of an infection and the severity of a subsequent disease.[56]

Host Response to Cryptosporidium

During Cryptosporidium infection, both innate and adaptive immune responses play critical roles in parasite clearance. Most of the information regarding host-pathogen interactions and immune responses to this protozoan parasite come from studies with Cryptosporidium-infected mice models. Many studies have reported the involvement of IECs in inflammatory response and parasite killing.[57],[58] IECs infected with C. parvum showed an elevated inflammatory response by upregulating numerous C, CC, and C-X-C classes of chemokine genes.[59] A recent study demonstrated that C. parvum infection suppresses transcription of the mir-424-503 gene through a nuclear factor-kappa B (NF-kB)-and histone deacetylases-dependent manner.[60] Another study suggests that C. parvum infection induces ileocecal adenocarcinoma and Wnt signaling in mice.[15] One member of the sirtuin family of proteins nicotine adenine dinucleotide (NAD)-dependent deacetylase sirtuin-1 (SIRT1), and an NAD-dependent deacetylase have been concerned in the regulation of multiple cellular processes, including inflammation, longevity, and metabolism. Recent gain and loss-of-function studies revealed that let-7i could modulate NF-κB activation through modification of SIRT1 protein expression these findings suggested a new role of let-7i in the regulation of NF-κB-mediated epithelial innate immune response.[61]

Cellular immune responses induced during Cryptosporidium infections are poorly understood. Various studies have reported the activation of toll-like receptors (TLRs) during Cryptosporidium infection leading to the release of various pro-inflammatory cytokines. The TLRs activate innate immune responses via NK-κβ pathway or by activation of caspase 1 inflammasome that induces proinflamatory cytokines, chemokines, and antimicrobial peptides.[62] TLR4 promotes C. parvum clearance in C. parvum infected mouse model.[63] In one such study involving C. parvum infected mouse, TLR4 was observed to be involved in promoting clearance of C. parvum.[64] In another study, Barakat et al. showed that mice deficient in B, T, and NK cells excrete higher oocysts concentration in comparison to mice that are deficient in B and T cells.[65] Higher levels of neutrophils, eosinophils, NK cells, and CD4 (+) CD25 T cells were found in C. parvum-infected BALB/C mice.[66]

Dendritic cells (DCs) play an important role in host immune responses during Cryptosporidium infection.[67] A few animal studies have reported the interaction between mouse DCs and C. parvum.[68] In one such study, it was observed that DCs expressed interferon-alpha (IFN-α) and IFN-β when exposed to live C. parvum sporozoites.[65] Bedi et al. studied the role of DCs using CD11c+ depleted mice model and observed a considerable increase in susceptibility to C. parvum infection.[67] Another study showed that CD103+ DCs are responsible for C. parvum clearance during infection.[69] In a study using human DCs stimulated with Cryptosporidium soluble and recombinant antigens, elevated secretion of Th-1 cytokines, specifically interleukin-12 (IL-12) p70, IL-2, IL-beta, and IL-6, was reported.[70] A few studies have also evaluated the role of CD+ T cells during Cryptosporidium infection; it was reported that CD4+ T cells significantly contribute toward the clearance of parasite.[71],[72] Human and mouse CD8+ T cells contribute significantly in the clearance of Cryptosporidium infection.[71],[72] The CD4+ T cells also provide a protective barrier against Cryptosporidium infection. In recent reports of immunodeficient mice infected with the parasite mounted parasite-specific serum IgG response as well as a systemic and mucosal IgA response; moreover, challenge infection led to a booster effect in immunoglobulin response despite the absence of oocyst shedding.[73] Non coding RNAs (ncRNAs) may modulate epithelial immune responses such as production of antimicrobial molecules and expression of cytokines during Cryptosporidium infection.[74]

Macrophages and neutrophils are important immune effector cells and contribute significantly to the intestinal inflammatory response during C. parvum infection.[75] These cells produce an important free radical called nitric oxide (NO) and NO synthesis is significantly increased in IECs that stimulate prostaglandin synthesis in the C. parvum-infected ileum.[76] A study on undernourished mice revealed that L-arginine plays a protective role in C. parvum infection, with involvement of arginase-I and NO synthase enzymatic actions.[77] One recent study also suggested that macrophages and IL-18 play prominent parts in NK cell–independent, IFN-γ–mediated innate immune pathway against C. parvum.[78]

Laboratory Diagnosis of Cryptosporidiosis

Various methods are available for the detection and diagnosis of Cryptosporidium as shown in [Table 1]. Most commonly used diagnostic methods involve detection of Cryptosporidium oocysts, antigens, or nucleic acid in stool samples. For routine examination, bright field microscopy is used along with Romanowsky stain, modified acid-fast staining, negative staining, or dimethyl sulfoxide modified acid-fast staining procedures.[79] Although these methods are low cost, they have low analytic sensitivity for detecting oocysts. On the other hand, direct and indirect immunofluorescent microscopy, although expensive, are more sensitive and oocysts are readily identified by this method. Various reference laboratories in the US and Europe use immunofluorescent microscopy as a gold standard for detection of Cryptosporidium.[80],[81] In the past decade, diagnostic interest has been greatly developed toward using Enzyme Immunoassays for detecting oocyst antigens, but still we are lacking clinic tools that can differentiate between oocysts of different species.[81] Rapid detection techniques such as immunochromatographic strip tests or immunochromatographic lateral flow tests are also available that show higher throughput with variable sensitivities (70%–100%).[82] A recent study suggested that the points-of-care tests for Cryptosporidium can be used as an alternative to conventional microscopy where diagnosis of this parasite is limited due to resource-poor settings.[83]

Table 1: Advantages and disadvantages of various techniques used for Cryptosporidium detection

Polymerase chain reaction (PCR) is widely used for genotyping Cryptosporidium, and a variety of PCR assays are available for the sensitive and accurate detection of Cryptosporidium species in clinical samples.[79] Amplification of Cryptosporidium genes encoding 18S rRNA is commonly used for this purpose. With the help of these assays, Cryptosporidium species identification and genotype differentiation can be done easily. Post-PCR analyses are usually based on the direct sequencing of the amplified products, or on the digestion with endonucleases followed by gel electrophoresis of the restriction fragments.[84] The conventional PCR approach does not provide information on pathogen viability and infectivity of pathogens; therefore, with the help of real-time PCR and inclusion/exclusion assays using vital dyes, we can get additional biologic information.[85] When needed, low amounts of Cryptosporidium oocyst DNA can be detected with the help of Loop mediated isothermal amplification (LAMP) PCR, and a recent study suggested that LAMP can be an effective and efficient way for conducting epidemiological studies of Cryptosporidium.[85]

Few studies have highlighted the importance of serological assays to detect IgG antibodies against cryptosporidium as an important tool for epidemiological studies.[16],[86] Antibody against Cp23 seems to correlate with distant infection, whereas responses to Cp17 suggest recent infections and antibody to P2 are associated with repeated infection.[87] There are numerous reports of in vitro cultivation techniques to grow Cryptosporidium in cell culture systems.[88] Different cell lines like MDCK, MA-104, HEP-2, Caco-2, AGS, HCT-8 can be used for this parasite culture and HCT-8 cell lines are the most compatible cell lines for Cryptosporidium growth.[89],[90] In spite of all these developments, it is still difficult to maintain Cryptosporidium in cell culture system that limits an in-depth understanding on various aspects of Cryptosporidium life cycles, host pathogen responses, and signaling pathways.

Treatment and Control Strategies

Present cryptosporidiosis treatments strategies are extremely limited. One promising treatment is nitazoxanide (NTZ), where a prospective randomized, placebo-controlled study showed that NTZ treatment reduced the duration of both diarrhea and oocyst shedding in patients.[91] Another randomized controlled trial of NTZ showed this drug is more effective in HIV-seronegative children in comparison to HIV-seropositive children.[92] Currently, NTZ is the only Food and Drug administration-approved drug for cryptosporidiosis that shows a moderate efficacy in immunocompromised individuals and children.[93] Other drugs including paromomycin, clarithomycin, azithromycin, rifaximin, rifabutin, and roxithromycinmay also be effective in inhibiting Cryptosporidium growth.[94],[95] Gargala et al. reported that Halogeno-Thiazolides (RM-5038) was more effective in comparison to NTZ in inhibiting the growth of Cryptosporidium in Mongolian gerbils.[96] Recent reports have also highlighted the effectiveness of pomegranate peel against C. parvum infection in a murine model.[97] More such studies are required to develop cheap and effective drugs to control Cryptosporidium. Nanotherapy-based approaches might be an effective drug delivery strategy, as a recent study showed Cryptosporidium-specific CP2 protein labeled NP-906 nanoparticles reduced the parasite level 200 folds in a cell culture model.[98]

Lack of long-term maintenance in cell culture and inability to carry out in vitro genetic modification in Cryptosporidium genome are hampering the development of new therapeutic strategies for Cryptosporidium. Various developmental stages of Cryptosporidium and its unique intracellular location, further complicates the drug development procedure. Recent emerging reports highlight the importance of protozoan kinases as a possible drug target for Cryptosporidium.[99]

Availability of full genome sequences of C. parvum and C. hominis has opened new avenues for developing effective drugs and vaccines for Cryptosporidium. In comparison to other apicomplexan parasites, the Cryptosporidium genome has undergone substantial gene lose and horizontal transfer; therefore, it contains highly streamlined metabolic pathways.[100],[101] Recently, using a novel in silico approach of reverse vaccinology, three new potential vaccine candidates Cp-15, Prolofin, and Cryptosporidium apyrase have been identified.[102] A DNA vaccine encoding Cp-12 and Cp-21 has shown protective immunity in BALB/c challenged with C. parvum.[103] Although no vaccines are commercially available for cryptosporidiosis yet, various experimental studies to develop attenuated vaccines and DNA vaccines are in progress.[104] At the present time, disease prevention efforts must focus on proper hygiene and clean sanitary conditions to minimize Cryptosporidium outbreaks, especially in developing countries where the prevalence is relatively high in human and animal populations.

Research Needs and Recommendations

As cryptosporidiosis is a zoonotic disease that spreads through contaminated food and water, epidemiological surveillance is warranted in regions with poor sanitation practice. More attention should be given toward the pathogen source tracking as a complementary tool using molecular studies to identify the actual human and animal contributor sources for infection transmission in a given geographic region. Microbial source tracking (MST) methods are helpful to discriminate between human and various nonhuman sources of fecal contamination.[105],[106] Recently, in one such MST study from India, it was observed that both human and animal sources are often present in the environment,[107] and contributing proportions of fecal loading can be estimated using quantitative PCR in combination with analytic modeling. In our recent study form rural India, we showed a high prevalence of Cryptosporidium in community water sources, humans, and domestic animals in the same geographical region.[108] Thus, MST is an active area of research and can be an effective method for source identification and characterization to address pathogen pollution and associated health risks.

The currently available diagnostic techniques for detection of Cryptosporidium have variable degrees of specificity and sensitivity; thereby none of these are unanimously recommended for routine laboratory diagnosis or in epidemiological screening studies. Therefore, it is now essential to develop more affordable diagnostic assays with high specificity and sensitivity for both medical as well as veterinary needs. Availability of such an assay would be boon to many developing countries with poor sanitation practice, where neonatal death due to Cryptosporidium-induced diarrhea is on the rise.